Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A and non-B hepatitis, with an estimated prevalence of 170 million cases (i.e., 2-3%) globally [Choo, et al., Science, 244: 359-362 (1989); Kuo, et al., Science, 244: 362-364 (1989); Purcell, FEMS Microbiology Reviews, 14: 181-192 (1994); Van der Poel, C. L., Current Studies in Hematology and Blood Transfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million individuals may be infected in the United States alone [Alter, and Mast, Gastroenterol. Clin. North Am., 23: 437-455 (1994)].
Upon first exposure to HCV only about 10% or less of infected individuals develop acute clinical hepatitis, while others appear to resolve the infection spontaneously. In the most instances, however, the virus establishes a chronic infection that persists for decades [Iwarson, FEMS Microbiology Reviews, 14: 201-204 (1994)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [Kew, FEM Microbiology Reviews, 14: 211-220 (1994); Saito, et al., Proc. Natl. Acad. Sci. USA 87:6547-6549 (1990)]. Currently, there are no broadly effective treatments for the debilitating progression of chronic HCV.
The HCV genome encodes a polyprotein of 3010-3033 amino acids [Choo, et al. Proc. Natl. Acad. Sci. USA, 88: 2451-2455 (1991); Kato, et al., Proc. Natl. Acad. Sci. USA, 87: 9524-9528 (1990); Takamizawa, et al., J. Virol., 65: 1105-113 (1991)]. The HCV nonstructural (NS) proteins provide catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [Bartenschlager, et al., J. Virol., 67: 3835-3844 (1993); Grakoui, et al. J. Virol, 67: 2832-2843 (1993); Grakoui, et al., J. Virol., 67: 1385-1395 (1993); Tomei, et al., J. Virol., 67: 4017-4026 (1993)].
Current therapies with alpha interferon alone and the combination of alpha interferon-ribavirin have been shown to be effective in a portion of patients with chronic HCV infection [Marcellin et al., Ann. Intern. Med. 127:875-881 (1997); Reichard et al., Lancet 351:83-87 (1998)]. Vaccine development has been hampered by the high degree of immune evasion and the lack of protection against reinfection, even with the same inoculum [Farci et al., Science 258: 135-140 (1992); Kao et al., J. Med. Virol. 50:303-308 (1996); Shimizu et al., J. Virol. 68:1494-1500 (1994); Wyatt et al., J. Virol. 72:1725-1730 (1998)]. Development of small molecule inhibitors directed against specific viral targets has thus become the focus of anti-HCV research. The determination of crystal structures for NS3 protease [Kim et al., Cell 87:343-355 (1996); Love et al., Cell 87:331-342 (1996); Yan et al., Protein Sci. 7:837-847 (1998)] and NS3 RNA helicase [Yao et al., Nat. Struct. Biol. 4:463-467 (1997)] has provided important structural insights for national design of specific inhibitors.
One key enzyme encoded by HCV is NS5B , which has been shown to be an RNA-dependent RNA polymerase (RdRp) [Al et al., Virus Res. 53: 141-149 (1998); Behreus et al., EMBO J. 15:12-22 (1996); DeFrancesco et al., Methods Enzymol. 275:58-67 (1996); Lohmann et al., J. Virol 71:8416-8428 (1997); Yuan et al., 1997, Biochem. Biophys. Res. Commun. 232:231-235 (1997); Ferrari et al., J. Virol. 73:1649-54 (1999)] NS5B is thus believed to be responsible for HCV genome replication. Cellular localization studies revealed that NS5B is membrane associated and distributed in the perinuclear region [Hwang et al., Annu. Rev. Biochem. 63:777-822 (1997)]. This coincides with the distribution of NS5A [Tanji et al., J. Virol. 69:1575-1581 (1995)], suggesting that NS5A and NS5B may stay together after proteolytic cleavage at the NS5A/NS5B junction. It has been postulated that the nonstructural proteins of HCV (NS3 to NS5B) may assemble into membrane-associated replication complexes that are competent for authentic RNA genome replication.
By itself, HCV NS5B RdRp appears to lack specificity for HCV RNA and can "copy back" heterologous nonviral RNA. This lack of specificity for HCV RNA may reflect the notion that additional viral or cellular factors are required for specific recognition of the replication signal, most likely present at the 3' untranslated region. Recent studies by Lohmann et al. (supra) demonstrated that NS5B alone can replicate the entire HCV genome via a copy-back mechanism initiated from the end of the 3' untranslated region.
The ability of recombinant NS5B from bovine viral diarrhea virus (BVDV) to initiate RNA synthesis by a primer-independent mechanism has recently been reported [Kao et al., Virology, 23, pp. 1-7 (1999)]. De novo initiation of RNA synthesis is likely to be the mode of initiation of BVDV replication in an infected cell. Kao et al. (supra) demonstrates that BVDV RdRp can preferentially initiate RNA synthesis by a de novo mechanism from short templates containing the signals for the initiation of genomic positive strand synthesis, and characterizes the requirements for the interaction between RdRp and the initiation site.
To date, there has been no report of de novo initiation of RNA synthesis by HCV NS5B.
Thus, there is a need in the art to resolve the current uncertainty concerning HCV genome replication.
There is a further need in the art to develop effective therapeutic strategies to inhibit viral specific features of HCV replicase.
The present invention addresses these and other needs in the art.